Biochip with maximization of the reactor number

ABSTRACT

This invention concerns a biochip, consisting primarily of one or several dieplates and one or several substrates with or without probe immobilized, and comprising a maximized number of reactors, wherein: a. said maximization of said reactor number is performed by minimizing structure-covered area on the substrate and/or maximizing effective area on the substrate, wherein said structure is partition structure of the reactor and/or structure other than the reactor; and b. said partition structure is characteristically based on surface partition, hydrophobic surface partition, or height-difference partition.

TECHNICAL SCOPE

This invention involves a biochip with a maximized number of reactors.The maximization is attained through maximizing the effective area onthe substrate, minimizing the area covered by partition structure on thesubstrate, or diminishing or even removing the substrate area covered bystructure other than reactors. Thereby the average substrate area perreactor is minimized, the reactor cost is reduced and the usageefficiency of the biochip is increased.

BACKGROUND TECHNIQUES

In this invention, term “biochip”, or “chip” refers to an analysisproduct, in which probes are immobilized on the surface of substrate inan addressable pattern, and are made to react specifically with targetmolecules of a biological sample under detectable conditions to obtainidentified result.

At present, the most prevailant biochip is peptides-chip and gene-chip.The peptides-chip is a biochip prepared by immobilizing sequences ofamino acids (including protein) on the substrate as probe. Gene-chip isprepared by hybridizing nucleic acid and/or nucleotide acid of a samplewith complementary target nucleic acid and/or nucleotide acid; or isprepared by combining with specific antibody so as to produce ahybridization result indicated through coloration reaction.

Biochip can be applied in extensive fields, including gene expressiondetection, gene screening, medicine screening, diagnosis and treatmentof diseases, inspection and protection of environment, identification injudicial affairs, etc.

The core of biochip is the reactor thereon. In this invention, the term“reactor” of biochip refers to the location and other relativestructures, in which probe array is immobilized and is made to reactspecifically with target molecules. The “probe” in this inventionincludes all bio-active substances that can be immobilized addressablyon the substrate, such as DNA, peptide, protein, cell, tissue etc. Inthis invention, the “substrate” refers to the component acting as solidmatrix to immobilize probe array in biochip; the “probe-plate” refers tothe substrate fixed with probe array. In this invention, biochip isdefined as mono-reactor biochip (n=1) and multi-reactor-biochip (n≧2),according to the number (n) of reactors on a biochip.

In this invention, the reactor is defined as flow-reactor ornon-flow-reactor according to whether the liquid media added can flowdirectionally in the reactor or not. The biochip characterized byflow-reactor and non-flow-reactor is defined as flow-biochip andnon-flow-biochip respectively.

In this invention, the reactor is defined as open-reactor or closedreactor according to whether the area above the probe array is open ornot in the whole process of detection. The biochip characterized byopen-reactor and closed reactor is defined as open biochip or closedbiochip respectively.

The biochip reactor, in most cases, simultaneously presents all thecharacteristics of the various reactors mentioned above. In thisinvention, this type of reactor is defined as a reactor with all of thesaid characteristics. The biochip characterized by this type of reactoris also defined in the same way. For example: when the area above theprobe array immobilized is open and the liquid media added can flow in acertain direction as required during the detection, this reactor isdefined as open-flow-reactor; the corresponding biochip is defined asopen flow biochip, or open flow chip in short; the rest may be deducedby analogy.

The current situation of biochip is as the following:

1. Non-Flow Biochip

Non-flow biochip includes closed non-flow biochip and open non-flowbiochip; the open non-flow biochip is most widely used recently.

The present open non-flow biochip is that with one open non-flowreactor, e.g. the biochip with activated microscope slide and probesspotted on the slide without any other structure added newly. Thisbiochip presents the following advantages: simple structure, easy andconvenient spotting and scanning, part of which can even be conducted inthe flowing medium propelled by external forces (e.g. liquid spray forwashing). However, this biochip brings about an high production anddetection cost and low efficiency when only a few types of probe arespotted and a small probe array is formed, e.g. peptide-chip orprotein-chip with a few types probes immobilized (e.g. several tohundreds). In this case, the practical use of this biochip is quitelimited.

The manufacturers and scientists have made many improvements on thistype of biochip in order to increase the efficiency, e.g. developingsingle open non-flow biochip into multiple open non-flow biochip. Atpresent, the multiple open non-flow biochip available in market is of abasic structure such as the following: a biochip with several to dozensof spherical or cubic open-reactors, in which the substrate is astandard substrate with the size of 25 mm×75 mm or 26 mm×75 mm(width×height), and partition structure height is less than 1.0 mm.Since the reactor of this biochip has merely inlet region and reactionregion, but no outlet region, the efficiency of this biochip is still tobe further improved, because the reactive media cannot flow as orientedin the reactor thus a continual operation is impossible. In addition,without outlet region, the height of partition structure is critical. Ifthe height is too low, it is easy to produce a cross contamination amongneighbor reactors; if the height is too high, it is impossible to readthe reaction result by the biochip scanner widely used today.

With regard to these problems, we invented <<a detachable biochip>>(Chinese patent application number 02113540.1). In the invention, thesubstrate with the probe arrays is assembled with device comprising thereactor partition structure high enough to prevent cross-contaminationamong the reactors in the reactions, while in the scanning it isdismantled from the device to suit the requirement of scanner.

Through all the biochips with multiple open reactors now in use, exceptthe removable biochip, present multiple reactors on one substrate, nosystemic effort has been made to maximize the number of reactors so far.e.g. other functional structures (e.g. the place for hand holding)except reactors have covered a large area on the substrate, usually over30%.

Another example for biochip with multiple closed non-flow reactor is<<micro-array biochip for multiple samples>> (Chinese patent applicationnumber 01112783.x). It presents multiple reactors without inlet oroutlet, and the probe array in the reactor is closed during thereaction, and is exposed in process except the reaction. The reactor isclosed with a polyester wafer after adding the sample, and disclosedafter the reaction for moving out the sample and washing the reactor.And then, the reactor is again closed after adding some other reagents,and again disclosed after the reaction, the process is repeated untilthe analysis is finished. It is very complex to use this biochip, whichis different from the characteristic biochip in this invention.

2. Flow Biochip

At present, the only flow biochip available is closed flow biochipincluding a capillary biochip device (Chinese patent number CN 2483395Y)and a micro-channel biochip. The micro-channel biochip is made throughpreparing micro-channel (width less than 0.05 mm and depth less than0.025 mm) on the substrate, and immobilizing the probe in the fluidicmicro-channel. When it is used, a sample is continuously added in themicro-channel with the probe immobilized, and then the reaction resultis read by the signal detective system. One example of the micro-channelbiochip is a biochip produced by Caliper Technologies Inc(www.caliper.com), in which the probe is immobilized in themicro-channel, formed by carving glass slide using laser techniques andetching techniques. In this biochip, there are several open reservoirwells connected with the micro-channel. The micro-channel biochippresents advantages of high sensitivity and a fast speed. However, itpresents some disadvantages: 1) a complex structure which make itsindustrial production difficile; 2) a specific flow condition whichneeds a sophisticated flow-rate controlled instrument, e.g. electronicosmosis device; 3) a specific condition for reading the reaction result,which needs other instrument than the common biochip scanner for somedetection like fluorescence label detection, because the probe isimmobilized in the interior surface of the micro-channel. One example ofthe capillary biochip device is a biochip consisting of a carry sheetglass (substrate) and several tiny poly-ethylamide gel strips bound onthe glass. The probe is immobilized on the strip which is used for theanalysis of target DNA, RNA and protein.

The maximization of the number of reactors on a biochip has not beenconsidered for present closed flow biochip.

In brief, all present biochips are principally directed to the object ofmaximizing the probe density in the reactor, in order to maximize thenumber of detected target molecules using a reactor. The probe densityhas been thousands and even ten thousands of probes per squarecentimeter, and a higher probe density is being developed. Thecorresponding spotter and scanner are also designed and manufactured forthis purpose. Althrough having some advantages, the above-mentionedbiochips present a great average substrate area per reactor. It is theaverage substrate area per reactor which determine the cost of biochip,in case of analyzing a sample with a few types of target molecules, e.g.less than 100 types. Only a few kinds of probe are needed for analyzinga few kinds of target molecule in a reactor. So, a pending problem inthe yield is to maximize number of reactor in a substrate within alimited area.

Invention Contents

So, this invention provides a biochip, consisting primarily of one orseveral dieplates and one or several substrates with or without probeimmobilized, and comprising a maximized number of reactor, wherein: saidmaximization of said reactor number is realized by minimizingstructure-covered area on the substrate and/or maximizing effective areaon the substrate, wherein said structure is partition structure of thereactor and/or structure other than the reactor; and said partitionstructure is characteristically based on surface partition, hydrophobicsurface partition, or height-difference partition.

In an embodiment, the subject invention provides a biochip in which saiddieplate and said substrate form, through connection, one or severalclosed flow reactors with inlet and outlet. In the biochip, saiddieplate and said substrate are connected either reversibly orirreversibly. The reversible connection, dis-connectable when desired,is performed with one or more following forces: a). mechanical forcegenerated by gravity, elasticity, screws or fixture; b). magnetic forcegenerated by magnet or electric magnet; c). removable adhesion forceproduced by adhesive; and which is. The dieplate is partial or entiremachine-eliminable when it is desired to open the top or/and to decreasethe height of said reactor formed by said irreversible connection.

In other embodiment, the subject invention provides a biochip in whichthe partition structure includes a concave structure. In the biochip,the concave structure contains one or more of the followingflow-controlling structures: a. hydrophilic material layer; b.hydrophobic material layer; c. layer of water-absorbing material basedon capillary actions; and d. leading ditches, leading trough, leadingstrip helpful for flow-controlling.

In another embodiment, the subject invention provides a biochip withmultiple reactors, in which the dieplate and the substrate are connectedby adhesion and form multiple open reactors with a partition structureof a height of more than 0.7 mm. In the biochip, as required, thepartition structure is either eliminable or height-deductible throughremoval of said adhesion or by mechanic action, wherein: a. the removalis performed with one or more following actions: a). physicochemicalaction of swelling and dissolving with water or/and organic solvents;b). physical action of ultrasonic wave; and c). mechanic action; and b.the mechanic action includes grinding, cutting, whittling or theircombination.

In another embodiment, the subject invention provides a biochip withmultiple reactors, in which a. the dieplate and the substrate form,through connection, multiple open reactors with special outlet region;and b. the partition structure on the dieplate presents a height of lessthan 1.0 mm and an hydrophobicity more than that of the substrate.

In the biochip with multiple reactors of this invention, the substratepresents a width of less than 20 mm when two or more lines of reactorsare formed on a substrate; or the substrate presents a width of lessthan 9 mm when only one line of reactors are formed on a substrate.

In the biochip with multiple reactors of the invention, the partitionstructure presents a height of more than that of parts or all of otherstructure on the biochip. The other structure includesscanning-reference-plane on the same plane as the substrate plane withimmobilized probes.

The biochip with multiple reactors according to this invention has anarea larger than that of the substrate, so parts or all of inletstructures or/and outlet structures of said reactor are set on dieplateregion where said dieplate goes beyond said substrate.

In the biochip with multiple reactors according to this invention, thereactor comprises inlet region and/or outlet region including one ormore of the following flow-controlling-structures: a. layer ofhydrophilic material; b. layer of hydrophobic material; c. layer ofwater-absorbent based on capillary actions; and d. leading ditch,leading trough, leading strip helpful for flow-controlling.

In the biochip according to this invention: the hydrophilic materialincludes: a). hydrophilic inorganic material including silicon, aluminumcompounds; b). hydrophilic organic material including polyacrylamidecompounds; c). hydrophilic coating; and d). natural macromolecularmaterial and its derivatives; the hydrophobic material includeshydrophobic organic material; and the water-absorbent includes: a).capillary, paper, membrane with hydrophilic surface; and b). poroussolid material with fiber or/and hydrophilic inorganic materials.

In an other embodiment, the subject invention is directed to a biochipwith two effective faces, in which the reactors with probe immobilizedare formed on both top surface and bottom surface of substrate, whereinthe structures on said top and on said bottom surfaces are mutuallyeither symmetrical or asymmetrical. In the biochip, the substratepresents a thickness more than 1.0±0.1 mm.

In the biochip according to this invention, said substrate is made fromany material which can form said reactor using a relatively smallaverage area, for instance inorganic materials such as glass, siliconand silicon compounds and etc., organic high polymer such aspolypropylene, polyvinylchloride, polystyrene, nylon and nitratecellulose, as well as organic materials whose surface is covered withmetals such as gold, silver or metal compounds.

The subject invention is directed also to a combined biochip, which iscomposed of several biochips above-mentioned, wherein: said severalbiochips are combined through insertion, adhesion and mechanicapposition; its total width is of no less than 25 mm; and the number ofsaid biochips combined is changeable as required.

FIGURE ILLUSTRATION

FIG. 1A is a top view of the dieplate of a biochip, comprising theclosed flow reactor and with a partition structure based on the surfacepartition, according to this invention. FIG. 1B is a bottom view of thedieplate in FIG. 1A. FIG. 1C is a schematic diagram of multiple probearrays on the substrate of the biochip in FIG. 1A. FIG. 1D is across-section cut straight along the line of a-a in FIG. 1A. FIG. 1E isa cross-section cut straight along the line of b-b in FIG. 1A.

FIG. 2 is a schematic diagram of how the dieplate in FIG. 1B and thesubstrate in FIG. 1C are connected by a magnetic fixturn.

FIG. 3A is a block diagram of a biochip according to this invention,comprising the reactor with the partition structure including concavestructure. FIG. 3B is a cross-section cut straight along horizontalplane of the biochip in FIG. 3A. FIG. 3C is a cross-section cut straightthrough horizontal plane of a biochip according to this invention,comprising the reactor with the partition structure including theconcave structure with water-absorbing layer.

FIG. 4A is a top view of a biochip according to this invention,comprising the multiple open reactors with the partition structure basedon the height difference. FIG. 4B is a cross-section of FIG. 4A cutstraight along the line of a-a. FIG. 4C is a schematic diagram of thebiochip in FIG. 4B after the height-deduction of the partitionstructure.

FIG. 5A is a top view of a biochip according to this invention,comprising the multiple open reactors with special outlet region andwith partition structure based on the higher hydrophobicity in partitionstructure than in the substrate. FIG. 5B is a cross-section of FIG. 5Acut straight along the line of a-a. FIG. 5C includes some schematicdiagrams of the outlet regions in varied shaped reactor in 5A.

FIG. 6A is a schematic diagram of the biochips of FIG. 4 and FIG. 5 withdifferent shaped reactor. FIG. 6B shows the possible shape of thereactors.

FIGS. 7A, 7B and 7C indicate some relationships between the substrateand the dieplate with reactor structure.

FIG. 8A is a cross-section of a biochip with two effective facesaccording to this invention. FIG. 8B is a schematic diagram of the probearrays immobilized on two surfaces of the substrate in the biochip ofFIG. 8A.

FIG. 9 is a schematic diagram of a biochip with two effective faces, inwhich the dieplate and the substrate are reversibly connected bymechanic fixture, according to this invention.

FIG. 10A is a top view of a biochip with two effective faces standing onits end, where the dieplate and the substrate are connected with eachother by reversible connection, according to this invention. FIG. 10B isa side-view of the biochip with two effective faces in FIG. 10A. FIG.10C is a bottom view of the biochip with two effective faces in FIG.10A.

DETAILED IMPLEMENTATION

In the development of a screening biochip for blood transfusion, we havecome to realize that how to improve the efficiency of the substrate is akey to cost controlling in the said biochip production. Only when thisproblem is solved can biochips with a better quality and a morecompetitive price than that of commonly used ELISA kit be available.Thus, “a biochip with a maximized number of reactors” has been selectedas a critical research subject in our study. Thereon researches on suchsubject as “a detachable biochip” (Chinese patent application number02113540.1), “a probe-plate containing closed reactor” (Chinese patentapplication number 02113864.8), “a flow biochip and its using method”(Chinese patent application number 02133622.9), “a biochip with severalfunctional surfaces” (Chinese patent application number 02113540.1), and“a biochip with small surfaced reactors” (Chinese patent applicationnumber 02134006.4) have been carried out successively. It is based onthese researches that the biochip in this invention comes into shape.

This invention aimed at maximizing the number of reactors per unit areaof substrate in biochip, namely to have as many reactors on onesubstrate within a limited area as possible, thereby to detect as manysamples as possible in one biochip.

In this invention, term “reactor” includes probe region, partitionregion, outlet region, and reaction well with or without inlet region.In the reactor, the probe region is a region where the probes areimmobilized; the reactor well is a location where added sample reactswith the probes; the outlet region is a region out of which liquidmedium and washing solution can flow after reaction; the partitionregion is a region surrounding reaction region, which serves to preventthe cross-contamination; the inlet region is a region through whichdifferent reaction media is added to reaction area, which may be anisolated region, or a part of probe region, outlet region or both. Theoutlet region can be on one side of reactor or in the region other thanprobe region, for instance, it may cover two sides or three sides of theprobe region or even all around the probe region. Outlet structure isthe structure in the outlet region, which can control the flow-rate ofreactive media, such as the layer of hydrophilic material, the layer ofhydrophobic material, the layer of water-absorbent material based oncapillary action as well as conductive ditch, trough, hole and striphelpful to flowing control.

The example for the outlet region on one side of probe region is anoutlet ditch on one side of reaction well; the example for outlet regionon all sides of probe region is a designed empty region in periphery ofprobe region, from which the liquid can be drained by ring-shapeddrainage pipe, when the partition region is higher than the proberegion; or from which the liquid can automatically flow to outlet regionthrough the peripheral probe region, when the partition region is lowerthan the probe region.

The hydrophilic material layer on the bottom of outlet trough mayaccelerate the discharge of the liquid from reaction well to outlet,which is helpful to retain liquid height in reaction well and avoidcross-contamination. The partition structure is a structure in thepartition region of a reactor, which is designed to hinder abnormal flowof the liquid so as to prevent the cross-contamination among reactors.Some examples are: the isolating ditch, the isolating line, theisolating trough, the layer of hydrophilic material and the layer ofhydrophobic material on the surface of the partition region. The layerof hydrophobic material on the surface of the partition region obstructsthe overflow from the reaction well in abnormal condition and keeps theliquid dots splashed on partition region from flowing, thereby itdiminishes the likelihood of cross-contamination. The layer ofhydrophilic material on the surface of partition region can alsodiminishes the likelihood of cross-contamination, by accelerating theliquid outflow in the reaction well and the partition region, when theheight of partition region is lower than that of probe plane.

This invention advocates a biochip, characterized by: a. its principalcomposition of one or more dieplates and one or more substrate orsubstrate conjugated with probe; b. its content of a maximized number ofreactors, wherein the maximization of the amount of reactors is carriedout by minimizing the substrate area occupied by reactor partitionstructure, by minimizing the substrate area occupied by other structuresexcept reactor and/or by maximizing the effective area of substrate; andits reactor partition structure based on surface partition, hydrophobicpartition or height difference partition.

In this invention, the “maximization of number of reactors” refers tomake formation of as many as possible reactors in a biochip; the“minimization of the substrate area occupied by reactor partitionstructure” refers to make the partition structure to occupy only aminimized partition region on the substrate, e.g. in the closed flowbiochip, in the biochip with concave partition structure, in the biochipwith multiple open reactors with special outlet region, and in thebiochip with multiple open reactors formed by adhesion, according tothis invention; the “minimization of the substrate area occupied byother structures except reactor” refers to make the other structuresexcept reactor to occupy only a minimized substrate area, e.g. in thebiochip with an biochip area greater that substrate area of thisinvention; “maximization of the effective area of substrate” refers tomake the effective area of substrate to be used as many as possible,e.g. the biochip with two effective faces according to this invention.

In this invention, the “partition” refers to separation of a reactorfrom all of other reactors in a same biochip; the “partition structure”refers to structure realizing the partition; the “surface partition”refers to the partition through one or more surfaces of piece used inthe biochip, e.g. the partition achieved by enclosing the reaction wellwith a cover piece (e.g. in the closed flow reactor of biochip of thisinvention), and by separating the reaction wells on the two surfaceswith substrate itself (e.g. in the biochip with two effective facesaccording to this invention); the “hydrophobic partition” refers to thepartition formed by employing hydrophobic material, e.g. the partitionin the biochip with multiple open reactors with special outlet region;the “height difference partition” refers to the partition achieved bymaking height difference between planes of the probe region andpartition region, e.g. the partition in the biochip with concavepartition structure and in the biochip with multiple open reactorsformed by adhesion in this invention.

To decrease the substrate area occupied by partition structure andthereby the average substrate area per reactor while guarantee theanti-cross-contamination capacity, the biochip of this invention is aclosed flow biochip with a partition structure characterized by thesurface partition. In the closed flow biochip in this implementationprogram, the dieplate is connected with substrate to produce severalclosed flow reactors with both inlet and outlet.

FIG. 1A is a top view of the dieplate of an example of the closed flowbiochip based on surface partition. FIG. 1B is a bottom view of thisdieplate. FIG. 1C is the schematic diagram of the probe array formed onsubstrate in this closed flow biochip. FIG. 1D is a cross-section cutstraight along the line of a-a in FIG. 1A. FIG. 1E is a cross-sectioncut along the line of b-b in FIG. 1A. The dieplate (2) comprises 8reactor cavities (5), corresponding to 8 probe arrays on the substrate(1). Every reactor cavity (5) has inlet (3) and outlet (4) for liquidsubstance. For example, the closed follow biochip can be formed byconnecting adhesively the substrate with the probe arrays and thedieplate with reactor cavity in FIG. 1B, inlet in FIG. 1A and FIG. 1D,and outlet in FIG. 1A and FIG. 1E. The biochip with the surfacepartition, in which the area of the partition regions is minimizedwithout the cross-contamination problem, presents a higher substrateefficiency.

The closed flow biochip in this invention is a variable biochip in whichthe reactor may be transformed from closed state to open state. In thebiochip, the dieplate and the substrate are connected by adhesive orother reversible connection manner. When the transformation needs toremove the reactor covering or/and reduce the reactor height, thedieplate and the substrate is dis-connected in case of the reversibleconnection, or the dieplate is partial or entire removed mechanically incase of the irreversible connection by irreversible adhesive. Forexample, the closed flow reactor of the biochip can be transformed fromthe close state before scanning, to the open state during the scanning,through dis-connecting the partition structure connected on thesubstrate. The variable biochip of this invention, in which the reactorcan be made the controlled transformation from close state to openstate, presents the following advantage: an expanded analytical scope,an increased assay speed, and a maximized number of reactors withguaranteed scanning quality.

In this invention, the connection between the dieplate and the substrateis attributable to one or more mechanisms as listed below: mechanicforce provided by gravity, elasticity, screw or fixture, magnetic forceby magnet or electric magnet, removable adhesion from adhesive. Forexample, to forming the closed flow biochip with several independentimpermeable reactors, a reversible connection between the dieplate andthe substrate can be achieved by the following mechanisms: the mechanicforce based on the gravity (through pressing the reactor component ontothe probe-plate with enough gravity); mechanic force based on the screw(through pressing the reactor component onto the probe-plate withscrewing); mechanic force based on the fixture (through pressing thereactor component onto probe-plate with coupling force between tenon andmortise); the magnetic force provided by magnet or electric magnet(through pressing the reactor component onto probe-plate with magnet orelectric magnet). The closed flow biochip in this invention, which cancarry out continual reaction and operation in the reactors, thus becomeshigh-efficiency.

FIG. 2 illustrates how the closed flow biochip in this invention isformed by magnetic device (or magnetic fixture): the magnetic deviceconsists of electric magnet 7 and plate 6 containing iron, which hasopening corresponding to the outlet of dieplate 2; the substrate 1 andthe dieplate 2 are clamped and reversibly connected with preciseorientation by the electric magnet 7, the plate 6 containing iron, andthe orientation notch 8. The connection can be dis-connected in the testprocess, as desired, without influencing detective precision. And thisfixture device can be used repeatedly.

To decrease the substrate area occupied by partition structure with alimited height while guaranteeing the anti-cross-contamination capacity,the biochip of this invention is an open biochip with partitionstructure including concave structure. In the biochip with concavepartition structure, the dieplate is connected with the substrate toform the convex and concave structure with a height difference. As shownin FIGS. 3A and 3B, this type of biochip is made through fixing one ormore substrates with probe array (probe-plate 11) on dieplate 12.

The concave partition structure of the biochip with multipleprobe-plates in this invention contains one or more of the followingflow-controlling structures: the layer of hydrophilic materials, thelayer of hydrophobic materials, the layer of water-absorbent materialsbased on capillary actions as well as conductive ditch, trough, stripadvantageous to flowing control. The said hydrophilic materials includeinorganic hydrophilic materials such as silicon, aluminum compounds andetc., organic hydrophilic materials like polyacrylamide compounds andetc, various hydrophilic coating, natural hydrophilic macromolecularmaterials as well as derivatives thereof; said hydrophobic materialsinclude many hydrophobic materials of organic compounds; saidwater-absorbent material includes any types of capillary, paper,membrane with hydrophilic surface, and solid porous materials with fiberor/and hydrophilic inorganic materials. As shown in 3C, theflow-controlling structure is the water-absorbent layer 13 amongsubstrates 11 and on dieplate 12.

To decrease the substrate area occupied by partition structure with alimited height in partial process while guarantee theanti-cross-contamination capacity, the biochip of this invention is abiochip with height-variable partition structure. In this biochip, themultiple open-reactors are formed by adhesive connection betweendieplate and substrate. In this biochip, the reactor partition structurewith an original height over 0.7 mm and preferably over 1 mm, based onthe height difference partition, is height-deductible. As shown in FIGS.4A and 4B, substrate 21 and dieplate 22 are adhered together to formreaction well 23 and partition structure 24 with a height over 0.7 mm,higher than that of the prevailing biochip in use. The partitionstructure is lowered or removed as desired through dis-adhesion or bymechanic action. The adhesive connection between dieplate and substratecan be relieved by the dis-adhesion, which is performed with one or moreof the following effects: physicochemical effect of the dissolving andswelling by water or/and organic solvents, physical effect of ultrasonicwave and mechanic force. Alcohol is preferred as the said organicsolvents. Or, as shown in FIG. 4C, the height of reactor, formed by thedieplate, can be reduced or removed by one or more kinds of mechanicforces like grinding, cutting, or whittling etc.

To decrease the substrate area occupied by partition structure with alimited height while guarantee the anti-cross-contamination capacity,the biochip of this invention is an open biochip with a partitionstructure characterized by surface hydrophobic partition. In thisbiochip, the dieplate and the substrate are connected to form multipleopen reactors with special outlet region, and the partition structure onthe dieplate presents a height of less than 1.0 mm and an hydrophobicitymore than that of said substrate. As shown in FIGS. 5A and 5 b, thesubstrate 31 and the dieplate 32 connected on the substrate form severalreactors 33. Since the hydrophobicity of dieplate 32 is higher than thatof substrate 31, it can better avoid cross-contamination among reactorsdue to surface tension. In addition, as shown in 5C, these reactorscontain various forms of the outlet region.

In the biochip schemed by FIG. 4 and FIG. 5, when two or more lines ofreactors are arranged on substrate, the width of substrate is less than20 mm, however when there is only one line of reactors on substrate, thewidth of substrate is less than 9 mm. As mentioned above, the biochip inFIG. 4 is one example of the height-variable biochip with multiple openreactors, and the biochip in FIG. 5, one example of the open biochipwith hydrophobic partition structure. In these cases, as shown in FIG.6, reactors can be rectangular, round or strip-shaped and etc.

In the biochips schemed by FIG. 4 and FIG. 5, the partition structure onthe biochip can be higher than substrate with probe, or even higher thansome or all other structures on biochip. Said other structures includescanning reference-plane that is located on the same plane as probeplane of substrate. As mentioned above, the biochip in FIG. 4 is oneexample of the height-variable biochip with multiple open reactors, andthe biochip in FIG. 5, one example of the open biochip with hydrophobicpartition structure. As showed in FIG. 7, the partition structure plane(the plane of dieplate) is the highest plane. diminishing or evenremoving the substrate area covered by structure other than reactors.

To decrease the substrate area occupied by structure other than reactionregion and thereby diminish the average substrate area per reactor whileguarantee the anti-cross-contamination capacity, the biochip of thisinvention is a biochip with a size larger than that of substrate. Thisbiochip with a larger size is the biochip with height-variable partitionstructure as schemed by FIG. 4, or the open biochip with a partitionstructure characterized by surface hydrophobic partition as schemed byFIG. 5. In the biochip, part or all of the inlet structure or/and outletstructure of reactor are located in the dieplate field beyond substrate.As shown in FIG. 7A and Example 4, the substrate 41 is connected withcentral part of the dieplate 42 with both side parts for scanningreference-plane 43, to form a biochip with biochip with a limited heightof hydrophobic partition structure (about 0.6 mm in the Example 4). Asshown in FIGS. 7B and 7C, the substrate 41 is connected with centralpart of the dieplate 42 to form a biochip, in which the reactor consistsof some structures such as the reaction well 44, the outlet ditch 45,the partition ditch 46 and the partition region 47. Among these reactorstructures, the outlet ditch 45 and the partition ditch 46 are formed onthe part of the dieplate 42, beyond the substrate 41.

In the biochips schemed by FIG. 4 and FIG. 5, its outlet region containsone or more of the following flow-controlling structures: the layer ofhydrophilic materials, the layer of hydrophobic materials, the layer ofwater-absorbing materials based on capillary actions as well asconductive ditch, trough, strip advantageous to flowing control. Saidhydrophilic materials include inorganic hydrophilic materials such assilicon, aluminum compounds and etc., organic hydrophilic materials likepolyacrylamide compounds, various hydrophilic coating, naturalhydrophilic macromolecular materials as well as derivatives thereof;said hydrophobic materials include many hydrophobic materials of organiccompounds; said water-absorbing material includes any types ofcapillary, paper, membrane with hydrophilic surface, and solid porousmaterials with fiber or/and hydrophilic inorganic materials.

To augment the effective area on the substrate while guarantee theanti-cross-contamination capacity, the biochip of this invention is abiochip with two effective faces, in which the probe is immobilized inreactors on top surface and bottom surface of substrate. The biochipstructures on these two surfaces are mutually symmetric or asymmetric.One or more probe arrays are immobilized on each of these two surfacesrespectively, to form, with corresponding structures, one or morereactors on each of these two surfaces respectively. These reactors maybe open-reactors or closed reactors, flow reactors or non-flow reactors.

FIG. 8A is the sectional diagram of an example of the biochip with twoeffective faces of this invention. FIG. 8B is the schematic diagram ofan example of probe arrays immobilized on two surfaces of substrate inthe biochip with two effective faces. FIG. 9 is the schematic diagram ofan example of the biochip with two effective faces in this invention, inwhich the biochip components are connected by reversible fixturemechanically. As shown in 8A, the biochip with two effective faces inthis invention is composed of two dieplates 52 and substrate 51 joinedby adhesive. The two surfaces of this substrate 51 are both conjugatedwith probe array (as shown in FIG. 8B). As shown in FIG. 9, the biochipis formed by two dieplates 52 and one substrate 51 forming sandwichstructure, and two pieces of fixture board 53 fixing said sandwichstructure on the position in fixation mould 54. The said fixture alsoincludes the corresponding magnetic fixture (as shown in FIG. 2), inaddition to the mechanic fixture.

The structures on top plane and the structure on bottom plane in thisinvented biochip with two effective faces are symmetric or asymmetric.For example, the reactors on a surface overlap, wholly or partially orwith nothing, to the projections from the reactors on another surface.All of the structures contained by a reactor are on one of the twosurfaces, or not on one of the two surfaces. As shown in 8A, thereactors made of one substrate 51 and two dieplates 52 are asymmetriceach other.

In this invented biochip with two effective faces, the thickness ofsubstrate is larger than 1.0±0.1 mm.

In this invented biochip with two effective faces, the partition amongreactors on any surface can employ partition mechanism described abovein this invention, or other partition mechanism.

As shown in FIGS. 10A-C, this invented biochip with two effective facesis composed of substrate 61 and two dieplates 62, and comprises severalreactors 65. In the course of sample detecting, the biochip with twoeffective faces will stand straight up. Reaction media are added frominlet 63, which under the action of gravity, will flow through reactor65 and flow out of outlet 64 directly. So it is much more convenient toconduct detection.

This invention also includes one kind of combined biochip. Itscharacteristic is: it is composed of several biochips as mentioned abovein this invention. These biochips are connected with each other by meansof insertion, adhesion and mechanic positioning; total width is no lessthan 25 mm; and the number of biochips or probe for said combining canbe changed according to requirements during the detection.

In this invented biochip, the material for substrate include all kindsof materials which can form biochip reactor with much small averagearea, for instance inorganic materials such as glass, silicon andsilicon compounds and etc., organic high polymer such as polypropylene,polyvinylchloride, polystyrene, nylon and nitrate cellulose, as well asorganic materials whose surface are covered with metals such as gold,silver or metal compounds.

The advantages of this invented biochip are: the chip cost used fortesting a sample is reduced by maximizing the number of reactors on unitarea of substrate when the testing desires only a few of probes.

IMPLEMENTATION EXAMPLES

All biochip substrates in this invention are substrates available inmarket or experimental substrates of SEDAC Company, France.

Implementation Example 1 A Flow Biochip with Covering-Variability Basedon Magnetic Force

The basic structure of biochip in this example is shown in FIG. 1 andFIG. 2. The dieplate with elastic material (2) is made from plasticplate containing iron with thickness of 1 cm. In the dieplate, there are8 reactor cavities 5 joined with inlet 3 and outlet 4. The inlet 3 canbe contacted closely with tip of pipette. The outlet 4 can be connectedwith inductive pipe. Probe arrays corresponding to reactor cavities 5are formed on the substrate 1. Besides, The surface of dieplate in touchwith substrate is stuck with one layer of rubber sealing spacer withthickness of 0.5 mm and without covering the reactor cavities. Themagnetic force fixture consists of electric magnet 7 below substrate 1and magnetic plate 6 with iron above dieplate 2. The orientation notch8, located between the magnetic force fixture and the dieplate 2, isused for fixing substrate 1. On substrate 1, according to cavityposition of dieplate 2, there are probe arrays immobilized in advancefor forming 8 reactors (their positions shown in FIG. 1C). The probesare HCV antigen, HIV₁₊₂ antigen and syphilis antigen, respectively. Theprobe array is a 3×3 probe array in which each kind of probe is spottedin 3 spots. The reactor partition is produced through running theelectric magnet, after dieplate 2, substrate 1 with probe immobilizedand magnetic force fixture are connected on position. Then theprocesses, such as sample addition, washing solution addition, themarker addition, and washing, are performed. The connection betweendieplate 2 and substrate 1 is removed by running off the electric magnetbefore scanning. The substrate 1 is obtained for the scanning.

In this implementation example, sample 1 is HCV antibody positive serum,sample 2 is HIV₁₊₂ antibody positive serum, sample 3 is syphilisantibody positive serum, sample 4 is positive control (the mixture ofHCV antibody, HIV₁₊₂ antibody and syphilis antibody positive serumcontrol), sample 5 is negative control (the HCV antibody, HIV₁₊₂antibody and syphilis antibody negative serum). All samples have beendetermined by pre-detection by classical mono-reactor open biochip underthe same reactive condition. The result is shown in table 1. TABLE 1 theresult of the biochip covered reversibly by magnetic force or flowbiochip HCV HIV1 + 2 sample antibody antibody syphilis antibody No 1 + −− No 2 − + − No 3 − − + No 4 + + + No 5 − − − control − − −

Implementation Example 2 A Open Biochip with Concave Partition Structure

The basic structure of biochip in this example is shown in FIG. 3. Eachsize of substrate 11 is 5.0×5.0×1.0 mm (length×width×height). Dieplate12 is a plastic plate with a size of 75×25×0.5 mm (length×width×height).Two rows eight columns totally 16 substrates 11 adhere to the dieplate12 with a rows distance of 5 mm and a columns distance of 4 mm. Theprobes, same as that in implementation example 1, are immobilized oneach substrate 11 in the form of the 3×3 probe array. A filter paperwith thickness of 0.5 mm, which presents 16 pre-open square holescorrespondent to the size and the distribution of substrate on thedieplate, is used as water-absorbing layer 13 to cover dieplate. Thus,the open biochip with concave partition structure including filter paperas the flow-controlling structure is formed (FIG. 3C). The reactivemedia are added directly on upper part of substrate 11. When addedenough quantity, the media flow to the filter paper in concavestructure. The filter paper delivers the liquid outward, and the liquidleaves from biochip through mechanic taking out or more water-absorbentmaterials. Before scanning, remove filter paper, then wash, andblast-dry biochip. The same result as that in implementation example 1is obtained using the biochip in this implementation example, when samesamples as that in implementation example 1 are subjected. In thescanning, the inlet rod of scanner descends 0.5 mm, compared with thatin scanning in implementation example 1.

Implementation Example 3 An Open Biochip with Height-Variable PartitionStructure, Which is Formed by Adhesion and is Lowered by MechanicalForce

The basic structure of biochip in this example is shown in FIG. 4. Thedieplate 22 is a plastic plate with an external size of 75×25×7 mm(length×width×height) and 16 round holes in the form of 2 rows 8columns. Each hole presents a diameter of 5 mm. The biochip is producedthrough adhering the dieplate 22 with the substrate 21, in which theprobes same as those in implementation example 1 are immobilized. In thetesting using this biochip, the samples are subjected in the reactorsusing a dispenser, and the washing buffer and the marker arerespectively subjected in the reactors and then removed from thereactors by a common ELISA plate washer. The samples are human serumsamples same as those in the implementation example 1, and the marker isthe goat anti-Ab against human immunoglobulin labeled with rhodanmine.The height of the partition structure on the dieplate is reduced from 7mm to that smaller than 0.5 mm by a cutter (as shown in FIGS. 4B and4C). After washed with distilled water and then alcohol, and dried witha blast-dryer, the biochip with the substrate and the dieplate remainderon the substrate is set in the scanner to analyze. The result obtainedby this analysis is the same as that in example 1.

Implementation Example 4 An Open Biochip with Hydrophobic PartitionStructure

The basic structure of biochip in this example is shown in FIG. 5.

The substrate 31 presents a size of 75.0×12.5×1.0 mm(length×width×height). The dieplate 32 presents an external size of75.0×25.0×1.6 mm (length×width×height). The central part of the dieplate32 is a concave groove with a size of 75.0×12.8×11.0 mm(length×width×height). The bottom of the concave groove presents, in theform of 2×8 array, 16 round holes with a diameter of 5 mm. The substrate31 is adhered to the concave groove of the dieplate 32 to form theprepared biochip with 16 wells. Probes used in this implementationexample are the same as FIG. 1 with 3×2 probes spots array. The size ofprobe region 34 in the central of reactive well is almost 1.2×1.0 mm.The area except probe region circled by the hole of dieplate is used asoutlet region (as shown in FIG. 5C). The biochip is blocked by bovinealbumin and ready for use. In this implementation example, one kind ofELISA plate washer is used which contains adding/pumping mechanism withconcentric circle, wherein adding is in the higher place of the centerand pumping is in periphery. The external circle of pumping can touch tothe outlet region of reactor where no probe is fixed on. The assayedhuman serum samples and detective method in this implementation are thesame as implementation 3. The detective results are the same too.

Implementation Example 5 A Biochip with Two Effective Faces

The basic structure of the biochip prepared in this Example is shown inFIG. 8A and FIG. 9. The connection among one substrate and two dieplatesfor forming this biochip is a reversible connection through fixture.

The substrate 51 presents a size of 75×25×1 mm (length×width×height).The two dieplates present a same size of 75×25×0.5 mm(length×width×thickness). There are 16 roles with a diameter of 4.5 mmon each of the dieplates, in the form of 2×8 array. The positions of theroles on a dieplate are asymmetric to those of the roles on anotherdieplate. The probes used in this implementation example are same asthose in implementation example 1. The probes are immobilized, in theform of 3×2 array, on the two surface of the substrate respectively. Theprobe arrays are located according to the positions of the roles in eachof the dieplates. After inactivated with bovine serum albumin and dried,the substrate is connected with the two dieplate. The fixture board 53and the fixation mould 54 are used for the connection with an accurateposition. The connection is achieved through forming two sealedsurfaces, one between the top surface of the substrate 51 and onedieplate 52 and another between the bottom surface of the substrate 51and another dieplate 52. Then it is ready for use. The human serum anddetective method used in this implementation example are the same asthose in implementation example 3. Before scanning, remove the press offixture board 53; take out the substrate, wash and blast-dry. Thescanning of the substrate is performed under the condition that inletrod of scanner descents 0.5 mm. The result obtained is same as that inimplementation example 1.

1. A biochip, consisting primarily of one or several dieplates and one or several substrates with or without probe immobilized, and comprising a maximized number of reactors, wherein: a. said maximization of reactor number is performed by minimizing structure-covered area on the substrate and/or maximizing effective area on the substrate, wherein said structure is partition structure of the reactor and/or structure other than the reactor; and b. said partition structure is characteristically based on surface partition, hydrophobic surface partition, or height-difference partition.
 2. The biochip of claim 1, wherein said dieplate and said substrate are connected to form one or several closed flow reactors with inlet and outlet.
 3. The biochip of claim 2, wherein: a. said connection between said dieplate and said substrate is either reversible or irreversible; b. said reversible connection, dis-connectable when desired, is performed with one or more following forces: a). mechanic force generated by gravity, elasticity, screws or fixture; b). magnetic force generated by magnet or electric magnet; c). removable adhesion force produced by adhesive; and c. said dieplate is partial or entire machine-eliminable, when it is desired to open or/and to lower height of said reactor formed by said irreversible connection.
 4. The biochip of claim 1, wherein said partition structure including concave structure.
 5. The biochip of claim 4, wherein said concave structure contains one or more of the following flow-controlling structures: a. hydrophilic material layer; b. hydrophobic material layer; c. layer of water-absorbing material based on capillary actions; and d. leading ditches, leading trough, leading strip helpful for flow-controlling.
 6. The biochip of claim 1, wherein: a. said dieplate and said substrate are connected by adhesion to form multiple open reactors; and b. said partition structure presents a height of more than 0.7 mm.
 7. The biochip of claim 6, wherein said partition-structure presents a height of more than 1.0 mm.
 8. The biochip of claim 6, wherein said partition structure is either eliminable or height-deductible through removal of said adhesion or by mechanic action, wherein: a. said removal is performed with one or more following actions: a). physical chemistry action of swelling and dissolving with water or/and organic solvents; b). physical action of ultrasonic wave; and c). mechanic action; b. said mechanic action includes grinding, cutting, whittling, or their combination.
 9. The biochip of claim 1, wherein: a. said dieplate and said substrate are connected to form multiple open reactors with special outlet region; and b. said partition structure is on the dieplate, wherein: a). said partition structure presents a height of less than 1.0 mm; and b). said partition structure is more hydrophobic than said substrate.
 10. The biochip of claim 6, wherein: a. said substrate presents a width of less than 20 mm when two or more rows of reactors are formed on a substrate; or b. said substrate presents a width of less than 9 mm when only one row of reactors are formed on a substrate.
 11. The biochip of claim 6, wherein said reactor is strip-shaped reactor.
 12. The multi-reactor-biochip of claim 6, wherein said partition structure presents a height of more than that of parts or all of other structure on the biochip.
 13. The multi-reactor-biochip of claim 12, wherein said other structure includes scanning-reference-plane on the same plane as the substrate plane with immobilized probes.
 14. The biochip of claim 6, wherein: a. the area of said biochip is bigger than that of said substrate; and b. parts or all of inlet structures or/and outlet structures of said reactor are set on dieplate region where said dieplate goes beyond said substrate.
 15. The multi-reactor-biochip of claim 6, wherein said reactor comprises inlet region and/or outlet region including one or more of the following flow-controlling-structures: a. hydrophilic material layer; b. hydrophobic material layer; c. layer of water-absorbent based on capillary actions; and d. leading ditch, leading trough, leading strip helpful for flow-controlling.
 16. The biochip of claim 5, wherein: a. said hydrophilic material includes: a). hydrophilic inorganic material including silicon, aluminum compounds; b). hydrophilic organic material including polyacrylamide compounds; c). hydrophilic coating; and d). natural macromolecular material and its derivatives; b. said hydrophobic material includes hydrophobic organic material; and c. said water-absorbent includes: a). capillary, paper, membrane with hydrophilic surface; and b). porous solid material with fiber or/and hydrophilic inorganic materials.
 17. The biochip of claim 1, a biochip with two effective faces, wherein: a. said probe is immobilized in said reactors on both top surface and bottom surface of substrate; and b. structures are symmetrical or asymmetrical on said top and bottom surfaces, mutually.
 18. The biochip of claim 17, wherein said substrate presents a thickness more than 1.0±0.1 mm.
 19. The biochip of claim 1, wherein said substrate is made of any material which can form said reactor with a relatively small average area, including: a. inorganic material including glass, silicon and silicon compound; b. organic macromolecular polymer including polypropylene, polyvinylchloride, polystyrene, nylon and nitrate cellulose; and c. organic material coated with metal including gold and silver.
 20. A combined biochip, composed of several said biochips of claim 1, wherein: a. said several biochips are combined through insertion, adhesion and mechanic apposition; b. its total width is of no less than 25 mm; and c. the number of said biochips combined is changeable as required. 